The biological activities of the NS1 protein of influenza A virus (NS1A protein) differ markedly from those of the NS1 protein of influenza B virus (NS1B protein). The NS1 proteins of these two genera of influenza viruses, which cause widespread human disease, likely mediate many of the viral countermeasures that combat cellular antiviral responses. Our overall aim is to identify all the activities of the NS1A and NS1B proteins and to elucidate how these different activities counter the cellular antiviral response and/or have other functions during virus replication. At early times after infection with either influenza A or B virus the transcription of cellular genes controlled by IFN (interferon)-stimulated response elements (ISREs) is activated without the involvement of IFNalpha/beta. Mature cellular mRNAs are produced in influenza B-, but not in influenza A-, infected cells. Our aims are to determine: (1) whether the absence of mature cellular mRNAs in influenza A virus-infected cells is caused by the block in 3' end processing of cellular pre-mRNA that is mediated by the NS1A protein, a function lacked by the NS1B protein; and (2) the mechanism by which ISREs of these cellular genes are activated in influenza A and B virus-infected cells. The NS1A protein contains a nuclear export signal (NES) which is activated between 2 and 4 hours post-infection. Our aim is to determine the mechanism of activation of this NES and its role during influenza A virus infection. The NS1B protein, but not the NS1A protein, binds ISG15, a human ubiquitin-like protein that is induced by either IFNalpha/beta or virus infection, and inhibits the conjugation of ISG15 to its target proteins. Because these protein targets have not yet been identified, it is not known why ISG15 conjugation would be detrimental to influenza B virus infection. Our goals are: (i) to establish how the conjugation of ISG15 to its target protein affects influenza B virus replication; and (ii) to identify the protein targets of ISG15 conjugation and to determine how this conjugation affects the activities of these target proteins. Other cellular proteins also likely bind to the NS1A and/or NS1B protein, e.g., the SRp54 protein that binds to NS1A, but not to NS1B, and our aim is to determine whether the binding of these other proteins has a functional role in influenza virus-infected cells. These functional analyzes of the NS1A and NS1B proteins will be complemented by structural studies using both nuclear magnetic resonance and X-ray crystallography.